5. Los Juicios de Núremberg: la consolidación de la interpretación simultánea
5.3 Un sistema de interpretación verdaderamente simultánea
Animals
The goats were identified by permanent horn markings:- Bashful (Ba), Doe (De), Dopey (Do), Grumpy (Gr), Happy (Ha), Max (Ma), Sleepy (Sl) and Sneezy (Sn). Each goat was dosed orally with five ml of 0.08% w /v solution of ivermectinl and then shorn. The bodyweig·hts of the ani mals ranged from 1 5 .0 to 2 1 .5 kg (mean 1 6. 8 8 ± 0 . 7 3 kg) at the beginning of the experi mental period and 1 7 .0 to 2 5 .0 kg (mean 2 0 .4 ± 1.0 7 kg) at the end.
All pharmacokinetic values are reported as the mean ± the standard error, unless otherwise specified.
Tissue cages
Silastic Medical-Grade Tubing (internal diameter 9.5 mm by external diameter 1 4 . 3 mm), was cut into eight centimetre long cylinders. Holes (0.2 m m dia meter) were punched through the walls o f t h e tubing, so that 40-5096 o f the surface area was perforated. Both ends of the cylinder were then sealed with medical grade silicone Type A glue2 (Chapt. 5, Plate 5 :2).
The tissue cages were thoroughly washed with a mild, non-oily soap and warm water. While still wet, they were pouched in sets of six into plastic envelopes, and autoclaved for 20 minutes at 1 2 1 °C.
After a two week quarantine period, five ani mals (Ba, De, Do, Gr and Ha) were starved for 1 2 hours but allowed access to water. General anaesthesia was induced in these ani mals using an intravenous solution of 5 96 thiopentone sodium3 administered at a dose rate of 25 mg/kg bodyw eight. Anaesthesia waB maintained through an endotracheal tube using 1 - 2 96 halothane.:!, in a circle anaesthetic system (fresh gas flow, 2 litres per minute).
1 . Ivomec ; Merk, Sharp &:: Dohme (NZ) Ltd., Wiri, New Zealand. 2 SilasticR Medical Adhesive A ; Dow Corning Corp. (NZ) Ltd. 3. Intraval sodiu mR: !\lay & Baker (NZ) Ltd., Lower Hutt. 4 FluothaneR ; ICI (NZ) Ltd., Wellington
The six surgical sites on each goat were clipped closely and cleansed using aqueous centri mide (1 %), then tincture of centri mide (40%), followed by povidone iodine ( 1 %) spray. A four centi metre anterioposterior incision was made in the skin, and a vertical subcutaneous pocket created by blunt dissection. A sterile silicone cage was inserted into each pocket. The wound was closed with two or three vertical mattress sutures using a non absorbable nylon suture m a terial5 , and then sprayed w ith povidone iodine (1 %) solution (C hapt. 5 , Plate 5 :3). The sutures were re moved 1 0 days following the surgery. The re maining three goats (i\Ia, Sl and Sn) were i mplanted si milarly, four weeks later.
Metronidazole
The metronidazole preparation for intravenous (IV) use was a 0.5% w/v aqueous solution (batch number P 7 97 )6. Any metronidazole solution re maining in an opened container at the end of each experi ment was discarded.
The concentrated product for intramuscular (Ii\I) use was presented as 20 grams of sterile, powdered metronidazole (T6 563, AHSO 2/8 5)7 , and a suspending agent (T6 5 6 1 , A HSO 2/8 5)8. Immediately before use, 3 0 ml of the suspending agent was added aseptically to 2 0 grams of the metronidazole powder, to produce a 40% w/v suspension. Vigorous shaking was necessary to prevent the powder from se ttling out.
All liquid metronidazole preparations were stored in the dark.
Drug administration
The ani mals were randomly assigned to one of two groups which received metronidazole, either by IV or IM injection. After a "wash out" period, the administration route for each ani mal was changed to the alternate option.
5 6 7 8
BraunamidR; B. Braun, Melsungen A , West Germany FlagylR; May & Baker (NZ) Ltd., Wellington
Metronidazole powder; May & Baker, Dagenham, England Suspending agent for metronidazole powder; !\lay & Baker, Dagenham, England.
7 7 Slow IV infusions of appropriate volumes of the 0.5% w/v metronidazole solution were made into the jugular vein through a 1 .5 c m long, 20 gauge needle. Either jugular vein was used, but care was taken to collect the first blood sample from the opposite side.
The concentrated 4 0 % w /v metronidazole suspension was administered by deep I!VI injection using a 1 . 5 c m long 1 8 gauge needle into the left cleido-occipital muscle, approxi mately 1 0 cm behind the ear. The injection site was then massaged as recommended by the manufacturers. The first blood sa mple follow ing the injection was always taken by puncture of the right jugular vein.
Samples of blood and tissue cage fluid
Collection
The time intervals for the collection of samples, for each experiment following IV or IlY! administration of metronidazole are shown (Table App. I:I).
The tissue cage fluid (0.5 to 3.0 ml) was removed by inserting a two cm long, 20 or 22 gauge needle, into the appropriate "cage", and then applying gentle suction to the attached six ml syringe.
The cages were sampled in a random order (C hapt. 5 , Fig. 5 : 1 ). Initially, if a capsule was missing from the site required, then a sample would be collected from the next cage in the order. For the subsequent experi ment, it was decided to sample each chamber only once every seventh sampling interval, to allow some time for equilibration of the cage fluid.
All samples containing metronidazole were protected from the light.
After 24 hours the blood and fibrin clots were removed, and the samples c e n t r i fuged at 1 7 3 2 0 for ten minutes. The supernatent was removed immediately and stored in aliquants at -20 ° C for up to 16 weeks.
Extraction
The frozen samples were thawed slowly, before chilled acetonitrile was added in a ratio of two parts of acetonitrile to one part of the sample. The acetonitrile/sample mixture was vortexed for one minute to ensure maxi mal mixing of the components, then centrifuged at 1 7 320 for 1 5 minutes. The
TABLE A pp. 1:1 Time intervals between the taking of blood or tissue cage fluid samples in goats following IV or IM injection of a 0.5% w/v
solution or a 40% w/v suspension respectively, of metronidazole.
Route of administration IV 0 . 5 % w /v metronidazole I I\ I 4 0 % w/v metronidazole Sample blood tissue cage fluid blood tissue cage fluid Time of sampling after administration (hours}
0, 0 . 2 5 , 0 . 5 1 , 2 , 3, 4, 5, 6 , 8 , 1 4 , 2 0 0, 0 . 2 5 , 0 5 , 1 , 2 , 3,4, 5, 6 , 7 , 8 , 1 0 , I 2,
I4, 24
0 , 0 . 5 , I , 2 , 3, 4, 5, 6 , 7, 8 , IO, 1 2 , 1 4 , 24 0, 0 . 5 , I , 2 , 3, 4, 5, 6 , 7 , 8 , I O , 1 2 , I 4, 24
7 9 supernatant w as removed from the protein precipitate, and i f the volume was sufficient, it w as filtered through a handheld 0 .5� Millipore filter9 . Samples thus prepared w ere stored at +4 °C in 1 . 5 ml glass vials1 0 , suitable for use in a Waters
wrspTl\:I
Model 7 1 0 . The samples were stored in screw capped vials, or analyzed immediately.Standard curve preparation
Pure metronidazole powder 8 8 2 3 RP (Ref. L O P 3 7 44)1 1 , was dissolved in normal goat serum ( 1 0 mg in 1 0 ml) by gentle agitation and war ming to 3 7 ° C . A dilution series of metronidazole concentrations, ranging from 50 mcg/ml to 0.1 mcg/ml, was prepared. These samples were processed for assay in the same manner used for the unknown samples. A standard curve was derived, from a plo t o f the magnitude of absorbance against the known metronidazole concentration.
Chromatography
The high pressure liquid chromatography unit consisted of a Waters Model 6 0 0 0 A Solvent D elivery1 2 system, a U 6 K injector and a Z- module radial compression separation system. The Z- module contained a Radial-PAK Cartridge (10)1 Bondapak C 1 8' 8 mm x 1 0 c m), and was set behind an inline filter and a guard column packed with the same material. A Waters programmable automator
(WrspT l\l)
Model 7 1 0 was connected into the system. The injection interval was set at 1 5 minutes and the injection volume at 50)11.
The mobile phase consisted of 7 5% aqueous potassium hydrogen phosphate (K H 2 P04) and 2 5 % methyl alcohol. The mixture was filtered1 3 (0.2 2)1 iVIillipore filter), and degassed under vacuum. The potassium hydrogen phosphate was made up to a concentration of one gram per litre, w ithout pH adjust ment (pH five to six). The running speed of the solvent was 1 .5 ml per minute.
9 l\1illiporeR Corp., Bedford, Massachusetts 0 1 730. 1 0 Waters Assoc.; Bedford, Massachusetts 0 1 7 57. 1 1 May & Baker; Dagenham, England.
1 2 . Waters Assoc.; Bedford, Massachusetts 0 1 7 5 7 . 1 3 MilliporeR Corp.; Bedford, Massachusetts 0 1 7 30.
A 4 5 0 variable w avelength detector w as set at either 0 . 0 1 or 0.04 absorbance units. The opti mum wavelength of 3 1 2 had been determined by a previously performed scan ( Cooper, pers. comm.) of the pure material. Initially the recording device was a Varian 5A chart recorder set at a chart speed of 10 cm per hour. This recording device was replaced w ith a Spectra Physics SP4270 Chromatography integrator, set at a chart speed of 0.2 cm per minute. Tissue cage removal
Approxi mately seven months after the cages had been inserted, a sample of fluid was aseptically withdrawn (approxi mately 0.5 to 3.0
ml),
from each cage. The majority of fluid w as placed in an individual vacutainer tube, and the remainder was used to make up a 'pool' in an EDTA vacutainer for each animal. Blood samples were collected for routine haemograms.The tissue cage sites were scrubbed with aqueous centrimide (1 %), followed by swabbing with tincture of centrimide (40 %). One to two ml of lignocaine hydrochloride (20 mg/ml)1 4 was infused about the most dependent point of the cage. After ten minutes, a two centi metre vertical incision was made through the desensitized skin and thick fibrous capsule surrounding the tissue cage. The cage was then grasped w ith forceps and removed. The wound w as sprayed with povidone iodine and left to heal (Chapt. 5, Plate 5 :4).
Calculations used for the determination of the disposition kinetics Intravenous injection
Following intravenous injection of metronidazole, the disposition data from individual goats were analyzed according to a one-compartment open model (Baggot, 1 97 4), w hich can be described by the monoexponential equation (equation 1 ). The line of best fit for the data w as calculated using a least squares regression analysis computer program 1 5 .
Where Cp =
Cp = Be-!3 t (1)
the plasma concentration at time t, (described in units of mcg/ml)
1 4 N opaine, Crown Chem. Comp., Lamberhurst, Kent, England. 1 5. Statistix, NH Analytials Software, USA.
B = p l a s m a d r u g concentration a t zero t i m e ,
extrapolated back from t h e elimination phase of the exponential curve.
a = the apparent first order disappearance rate
constant, obtained from the slope of the excretion phase of the se milogarithmic plot of serum drug concentration versus time. This constant is described in units of reciprocal time (per min. or per hour).
e = the base of the natural logarithm (ln).
8 1
The
q
is the time taken for the plasma concentration of a drug to be reduced by 5 0 96. An esti mate of theq
of a drug may be obtained directly from the beta (also termed eli mination, or excretion, or terminal) phase, of the se milogarithmic plot of plasma drug concentration versus time, following intravenous (IV) administration of a single dose of a drug (Fig. App. I:1). Theq
is described in units of either minutes or hours.The elimination rate constant, B , was calculated by employing equation 2
(Baggot, 1 97 7). tt = In 2 = a 0 . 6 93 a (2)
The zeroti me concentration intercept of the eli mination phase (B) was obtained by the back extrapolation of the terminal linear segment of the semilogarithmic plot (Fig. App. I:l).
F or the raw data of two goats, in w hich distinct distribution phases indicated consistency w ith a two-compart ment open model, the disposition kinetic values were calculated according to a biexponential expression ( Baggot, 1 97 4) (equation 3).
Where Cp, B, e, B and t are as defined in equation 1 . A = t h e p l a s m a d r u g c o ncentration
i n t e r c e p t at z e r o t i me of the distribution phase of the disposition curve,
ex = the appar ent first order disappearance rate
constant, obtained fro m t h e slope of t h e distribution phase, o f the se milogarithmic plot of plasma drug concentration versus time.
The least squares regression line describing the distribution phase was obtained by the method of residuals
(
Sams, 1 98 4). The beta phase points, previously deter mined by extrapolation of the terminal linear segment back to time zero, are subtracted from the actual data points throughout the curve. The calculated differences are called residual concentration-time values, w hich are then plotted on the semilogarithmic graph of serum metronidazole concentration against time(
Baggot, 1 9 7 7 ).The negative slope of this line yields ex , and its zero time intercept is A
(
Baggot, 1 9 7 7 ).The volume of distribution was calculated by dividing the administered dose
(D)
by the product of the eli mination rate constant(
B ) and the area under the concentration-time curve(
A UC), according to the following(
equation 4).V d
(
area)
=D
S . AUC
(4)The area under the concentration-time curve (AUC) representing the total amount of drug which appears in the serum after administration, was calculated by the trapezoidal method using values obtained directly from the graph
(
Baggot, 1 97 8 )(Fig. App. 1:2).
8 3
Fig . App . 1 : 1 Semilogarit hmic plo t of p l a sm a drug concen
t ration ver sus time f ollowing administ ratio n
o f a single in t ravenous d o se . Graphical
t echnique for estimating t he half-life of
a drug is illust r a t ed (fr o m B aggo t , J . D .
[ 1 9 7 7 ] ) : of Disp o sition in Domest ic Animals . P ub l . by S aunder s Comp . , L ondon .
Fig . App . 1 : 2 Diagrammat ic represent a t io n of the t ra pezoid al met hod used t o cal culate t he area under t he pla sma concen t ra t io n time curve
(AUC ) . (From B aggot , J . D . [ 1 9 7 8 ] ) :
9 8 7 \ \ \